Mammalian voltage-gated sodium channels are pore-forming membrane proteins responsible for the initiation and propagation of action potentials in excitable membranes in nerve, skeletal muscle and heart cells. The controlled gating of sodium channels in response to membrane depolarizations is necessary for normal electrical signaling and establishing of intercellular communication. Voltage-gated Na+ ion channels consist of one large α-subunit (about 200 kDa) and one or two smaller β-subunits. The α-subunits are designated “Nay” (Na for sodium channel and v for voltage-gated), followed by a numbering system for the particular isoform. The Na+ channel α-subunit isoforms contain four homologous repeated domains (D1–D4) each with six transmembrane segments (S1–S6). The α-subunit protein alone forms a functional channel when expressed in mammalian expression systems. The four repeated domains are hypothesized to assemble as a pseudotetrameric structure with the permeation pathway situated at the center. FIG. 1 is a cartoon depicting one conceptualization of how the Nay protein arranges itself with respect to the membrane. The cartoon is not accurate; it is an expanded model that does not attempt to depict how the four S6 segments come together to form the sodium channel, but it facilitates an understanding of how the proteins might align with respect to the inside and outside of the excitable membrane. In fact, recent studies suggest that four S6 C-termini may jointly close the voltage-gated cation channel at the cytoplasmic side, probably as an inverted teepee structure.
Several pieces of evidence suggest that S6 segments are involved in Na+ channel gating. First, a number of receptors for various therapeutic drugs and neurotoxins such as local anesthetics (LAs), antiarrhythmics, anticonvulsants, antidepressants, pyrethroid insecticides, batrachotoxin (BTX), and veratridine, are situated at the middle of multiple S6 segments. Upon binding, these ligands exert their pharmacological actions on the Na+ channel, presumably in part via their corresponding S6 receptor. In particular, BTX drastically modifies Na+ channel activation, fast inactivation, and slow inactivation, suggesting that its receptor is linked to these gating processes.
The invention herein described arose from a hypothesis that S6 segments may be structurally geared for channel activation by lateral/rotational movement via a flexible gating hinge, a glycine or serine residue located at the middle of the inner Na+ channel S6 segments. This gating hinge could have two different conformations. One is in its relaxed straight α-helical form, which closes the channel at the S6 C-terminal end, and the other is the bendable α-helical form, which may bend outward at a 30° angle and thus splay open the channel at the S6 constricted C-terminus. After channel activation, S6 segments may then form the docking site for the fast-inactivation gate. A putative Na+ channel inactivation gate has been delineated at the intracellular linker between D3 and D4 by West et al. [Proc. Natl. Acad. Sci. USA 89:10910–10914 (1992)]. This linker could be situated at the C-termini of S6 segments, where the inactivation gate may plug the open channel while it binds to its docking site. This plugging mechanism has recently been demonstrated in voltage-gated K+ channels [Zhou et al., Nature 411:657–661 (2001)]. The foregoing hypothesis is useful because it provides a framework for interpreting the results and making predictions. However, it is important to note that the invention is based on the results, not the hypothesis, and the hypothesis should not be viewed as a limitation on the claimed invention.
There is very close homology among the S6 segments of mammalian Nav proteins so far identified. This homology extends both through species and through isoforms of the Nav protein. As can be seen in the comparison below, the few variations that exist among the amino acids in the amino terminal portion of the S6 segments are very conservative replacements, and the carboxy terminal 11 amino acids of the S6 segments of all four domains are identical for rats and humans for both of the muscle sodium channel proteins Nav1.4 and Nav1.5:
D1S6 1611162126humanNav1.1YMIFFVLVIFLGSFYLINLILAVVAMAY(SEQ ID NO.: 1) Nav1.2YMIFFVLVIFLGSFYLINLILAVVAMAY(SEQ ID NO.: 2) Nav1.3YMIFFVLVIFLGSFYLINLILAVVAMAY(SEQ ID NO.: 3) Nav1.4YMIFFVVIIFLGSFYLINLILAVVAMAY(SEQ ID NO.: 4) Nav1.5YMIFFMLVIFLGSFYLVNLILAVVAMAY(SEQ ID NO.: 5) Nav1.8YMIFFvVvIFLGSFYLVNLILAVVAMAY(SEQ ID NO.: 6) Nav1.9YMIFFVVVIFLGSFYLINLILAVVAMAY(SEQ ID NO.: 7) ratNav1.4YMIFFVVIIFLGSFYLINLILAVVAMAY(SEQ ID NO.: 8) Nav1.5YMIFFMLVIFLGSFYLVNLILAVVAMAY(SEQ ID NO.: 9) Nav1.6YMIFFMLVIFVGSFYPVNLILAVVAMAY(SEQ ID NO.: 10) Nav1.7YMVFFVVVIFLGSFYLVNLILAVVAMAY(SEQ ID NO.: 11) Nav1.8YMVFFMLVIFLGSFYLVNLILAVVAMAY(SEQ ID NO.: 12) D2S6 1611162126humanNav1.1CLTVFMMVMVIGNLVVLNLFLALLLSSF(SEQ ID NO.: 13) Nav1.2CLTVFMMVMVIGNLVVLNLFLALLLSSF(SEQ ID NO.: 14) Nav1.3CLIVFMLVMVIGNLVVLNLFLALLLSSF(SEQ ID NO.: 15) Nav1.5CLLVFLLVMVIGNLVVLNLFLALLLSSF(SEQ ID NO.: 16) ratNav1.4CLTVFLMVMVIGNLVVLNLFLALLLSSF(SEQ ID NO.: 17) Nav1.5CLLVFLLVMVIGNLVVLNLFLALLLSSF(SEQ ID NO.: 18) D3S6 1611162126humanNav1.1MYLYFVIFIIFGSFFTLNLFIGVIIDNF(SEQ ID NO.: 19) Nav1.2MYLYFVIFIIFGSFFTLNLFIGVIIDNF(SEQ ID NO.: 20) Nav1.3MYLYFVIFIIFGSFFTLNLFIGVIIDNF(SEQ ID NO.: 21) Nav1.4MYLYFVIFIIFGSFFTLNLFIGVIIDNF(SEQ ID NO.: 22) Nav1.5MYIYFVIFIIFGSFFTLNLFIGVIIDNF(SEQ ID NO.: 23) Nav1.8MYLYFVIFIIGGSFFTLNLFVGVIIDNF(SEQ ID NO.: 24) ratNav1.4MYLYFVIFIIFGSFFTLNLFIGVIIDNF(SEQ ID NO.: 25) Nav1.5MYIYFVVFIIFGSFFTLNLFIGVIIDNF(SEQ ID NO.: 26) Nav1.7MYLYFVVFIIFGSFFTLNLFIGVIIDNF(SEQ ID NO.: 27) Nav1.8MYIYFVVFIIFGGFFTLNLFVGVIIDNF(SEQ ID NO.: 28) D4S6 1611162126humanNav1.1GIFFFVSYIIISFLVVVNMYIAVILENF(SEQ ID NO.: 29) Nav1.2GIFFFVSYIIISFLVVVNMYIAVILENF(SEQ ID NO.: 30) Nav1.3GIFFFVSYIIISFLVVVNMYIAVILENF(SEQ ID NO.: 31) Nav1.4GICFFCSYIIISFLIVVNMYIAIILENF(SEQ ID NO.: 32) Nav1.5GILFFTTYIIISFLIVVNMYIAIILENF(SEQ ID NO.: 33) ratNav1.4GICFFCSYIIISFLIVVNMYIAIILENF(SEQ ID NO.: 34) Nav1.5GILFFTTYIIISFLIVVNMYIAIILENF(SEQ ID NO.: 35)
Except for a single I→V change at position 7 of D3S6, the rat and human Nav1.4 and Nav1.5 sequences are identical for all four S6 segments. Because of the very high degree of conservation (in fact identity) of the 11 amino acids at the carboxy termini of the S6 segments, the person of skill in the art expects that substitution in this region will have the same effect on sodium channel function across mammalian species and across isoforms of the Nav1 protein.
The numbering shown in the charts above is the standard numbering used to identify the 28 amino acids in the S6 segments by their position within that segment. A separate system of numbering that may be applied to those same amino acids derives from their position within the sequence of the whole protein. Because the amino acid sequences of members of the Nav family of proteins vary widely outside the transmembrane regions, the protein sequence residue numbers assigned to the corresponding amino acids in the S6 segments differs among species and among sodium channel protein isoforms within species. Thus, the leucine identified as residue 19 in segment 6 in domain 1 (D1S6) is L407 in human Nav1.5, L408 in rat Nav1.5, L441 in human Nav1.4 and L435 in rat Nav1.4. Similarly, the isoleucine identified as residue 23 in segment 6 in domain 4 (D4S6) is I1770 in human Nav1.5, I1771 in rat Nav1.5, I1581 in human Nav1.4 and I1589 in rat Nav1.4. Unless otherwise noted, amino acids will be identified hereinafter, when referring to the whole protein, according to their position in rNav1.4. Thus A438 refers to the alanine that occurs at position 438 in rNav1.4.